(31b) Designing Hydrogel Scaffolds to Direct Cell Fate and Behavior

Introduction: The current state-of-the-art of bioprinting involves spatially patterning primary or differentiated cell types within a 3D hydrogel microenvironment. There is a desire for bioprinting to be used to similarly pattern naive stem cells to engineer 3D environments for studying stem cell behavior and expansion in three-dimensional culture as well for studying differentiation and self-organization into precisely designed functional tissues or organoids. We hypothesized that we could develop a hydrogel for encapsulating stem cells that enables the stem cells to proliferate while maintaining their stemness. We have developed a viable method to pattern encapsulated human induced pluripotent stem (hiPS) cells within a poly(ethylene glycol)-based hydrogel using digital light projection (DLP)-based bioprinting.

Materials and Methods: The bioink was optimized for stem cell adhesion and stemness maintenance. 8-arm PEG-norbornene (M_n = 40 kDa) was chosen as the primary matrix component as it can maintain a three-dimensional structure at low stiffness (i.e., less than 1 kPa). A laminin integrin binding motif, YIGSR, was incorporated in the bioink to enable cell adhesion. A matrix metalloproteinase (MMP) degradable crosslinker was used to finely tune the rate of matrix remodeling. The highly cytocompatible photoinitiator lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP) was also incorporated at a concentration of 0.25% w/v. hiPS cells were obtained from WiCell (IMR90-4 cell line). When dissociating the hiPS cells for bioprinting, we optimized the protocol to achieve embryoid bodies of approximately 4-6 cells. All bioprinting was done on our custom-designed DLP-based 3D printing system, using a 365-nm LED light source. Local stiffness was characterized via nanoindentation (Piuma, Optics11 Life). Fluorescence imaging was done on either a Leica DMI6000 B inverted microscope or on a Leica SP8 confocal microscope. Immunofluorescence staining was performed to characterize stem cell (Oct4, Nanog, Sox2) and proliferation (Ki-67) markers.

Results and Discussion: We optimized the bioink formulation for encapsulating stem cells that can stably maintain their stemness by developing a resultant hydrogel with a stiffness of 500 Pa and that has integrin binding sites preferred by stem cells. We characterized the distribution of the conjugated YIGSR peptide within a hydrogel construct by using a fluorescently tagged YIGSR peptide. We found that as expected the peptide was homogenously distributed within the hydrogel volume. We also confirmed that the addition of the YIGSR peptide did not affect mechanical properties of the hydrogel versus control. We bioprinted the hiPS cells to characterize their stemness and proliferation over the course of 7 days. On Day 7, the stem cell markers were still highly expressed, and the proliferation marker fluorescence signal density had increased. Notably, we did not need to add the ROCK inhibitor small molecule Y-27632 to achieve these results. To our knowledge, the only other method that doesn’t require Y-27632 during passaging of stem cells is the commercial product iMatrix, which is recombinant laminin-511. To further demonstrate the viability and function of the 3D-cultured bioprinted hiPS cells, we passaged the cells by degrading the matrix with collagenase and spinning down the cell-laden degraded hydrogel. We then seeded the cells with iMatrix in a 6-well plate and observed hiPS cell colony formation and proliferation.

Conclusions:

Our results indicate that we have developed a hydrogel formulation compatible with light-based bioprinting to encapsulate human stem cells in a 3D matrix that enables stemness maintenance and proliferation. This work can immediately enable bioreactor scale up of stem cell production. Additionally, the ability to 3D pattern stem cells could enable the bottom-up engineering of multicellular tissue constructs.